Isolation and Characterisation of Phage-Displayed Scfv Antibodies Targeting PfHSP70 and PfLDH of Plasmodium Falciparum

Plasmodium falciparum is the most virulent human malaria parasite and is responsible for numerous deaths annually. The increasing resistance of P. falciparum to antimalarial drugs necessitates the development of improved diagnostic tools for timely malaria detection. Malaria biomarkers such as PfHSP70 and PfLDH are highly valuable for malaria detection because they are essential for parasite survival and are consistently expressed during infection. PfHSP70 is associated with the parasite's stress adaptation and proteostasis mechanisms under febrile and drug-induced conditions, while PfLDH plays a central role in glycolytic metabolism and redox balance. Their functional importance, parasite specificity and elevated expression during active infection make these proteins reliable molecular indicators for the sensitive and specific detection of P. falciparum, thereby supporting their potential application in rapid diagnostic and biosensing platforms for malaria surveillance and disease management. In this study, an M13 phage-displayed single-chain variable fragment (scFv) antibody library was used to screen for antibodies against recombinant PfHSP70 and PfLDH. Four rounds of biopanning were conducted to enrich high-affinity binders, followed by ELISA, UV-visible spectroscopy and microscale thermophoresis (MST) to evaluate specificity and binding affinity. Functional interactions were assessed in Escherichia coli expressing PfHSP70 or PfLDH. Recombinant PfHSP70 and PfLDH were successfully expressed and purified from E. coli, with approximately 50% of selected colonies demonstrating significant binding to both targets, confirming the enrichment of antigen-specific phages. Specific phages were validated using ELISA and transmission electron microscopy (TEM). Selected scFvs exhibited strong target binding, with MST-determined dissociation constants (Kd) of 7.47 μM for PfHSP70 and 3.64 μM for PfLDH. Exposure to scFv-displaying phages impaired the survival of PfHSP70- and PfLDH-expressing E. coli and induced spectral shifts consistent with protein-antibody interactions. This study validates phage display as a robust platform for isolating high-affinity scFvs against P. falciparum targets. These binders have the potential to develop into low-cost diagnostic tools, addressing the urgent need for novel diagnostic interventions. Nevertheless, further studies are required to confirm binding specificity and assess translational applicability.